Method of manufacturing dual-specific T-cells for use in cancer immunotherapy

ABSTRACT

The present invention relates to autologous dual-specific lymphocytes, methods of making and uses for the treatment of tumors. In particular, the invention relates to methods producing autologous dual-specific lymphocytes comprising an endogenous receptor for at least one tumor associated antigen and an exogenous receptor for a strong antigen.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application represents the national stage entry of PCTInternational Application No. PCT/US2016/062831 filed on Nov. 18, 2016and claims the benefit of U.S. Provisional Patent Application Ser. No.62/257,429 filed on Nov. 19, 2015 entitled “Method of Manufacturing DualSpecific T-Cells for Use in Cancer Immunotherapy,” the contents of whichare incorporated by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

N/A

BACKGROUND OF THE INVENTION

The field of the invention is cancer immunotherapy. More particularly,the invention relates to adoptive cell transfer for the treatment ofcancer.

Various types of cancer are often refractory to standard treatments suchas chemotherapy or radiation. Adoptive cell transfer (ACT) ofgenetically engineered T cells has become a promising cancerimmunotherapy for hematologic malignancies (1-4). This approach includesengineering and expanding the tumor-infiltrating lymphocytes, which canrecognize tumor associated antigens (TAAs), followed by infusing theminto patients to induce a tumor specific immune response. Despite therecent success in treating hematopoietic malignancies (Maus, et al.),the efficacy of such an approach is curtailed when treating solid tumors(2,5,6). The primary hurdles that must be overcome for ACT to beeffective against solid tumors include: inadequate responses ofadoptively transferred T cells, especially in dealing with heterogeneouscancerous cells that bear a wide range of tumor associated antigens(2,6); reduced migration of adoptively transferred T cells into thetumor (7); and, the immunosuppressive microenvironment within tumorsthat often induces a rapid loss of T cell effector function (8).

Using infectious pathogens that stimulate a patient's immune system andbreak immunosuppression in the tumor microenvironment is a century oldstrategy that is now being rejuvenated to enhance cancer immunotherapy(9). Bacillus Calmette-Guérin (BCG), a live attenuated strain ofMycobacterium bovis, has been widely used in treating bladder cancer andmelanoma for decades (10,11). Although effective, BCG only inducestransient and non-specific antitumor immune responses. One reason forthis is that the inflammatory reaction induced by BCG does not targetand immune response to the tumor. To generate a tumor-specific T cellresponse, recombinant Listeria monocytogenes (LM) expressing engineeredTAAs have recently been developed and shown promising results intreating multiple cancers, including breast and pancreas (9). Owing tothe heterogeneity of tumor cells, it remains challenging for recombinantLM-based immunotherapies targeting a single TAA to provide durable andcomplete regression of cancer because cancer cells that don't expressthe targeted TAA are able to evade immunosurveillance (2,6,7,9). Thus,there is a critical need for new strategies that generate robust T cellresponses with broad coverage of tumor antigens to improvepathogen-based cancer vaccines.

SUMMARY OF THE INVENTION

The present invention overcomes the aforementioned drawbacks of ACT byproviding an innovative approach that not only overcomesimmunosuppression, but also recruits robust polyclonal anti-tumor T cellresponses to the very site of the tumor. First, autologous lymphocytes(e.g. CD8+ T cells) taken from a patient which are specific to aplurality of tumor associated antigens are genetically engineered torecognize both the tumor associated antigen and a strong antigen that isnon-tumor in vitro. These engineered autologous lymphocytes are thentransfused back into the patient in combination with injecting orinstilling the strong antigen directly into or to the tumor. Thedual-specific CD8 T cells expand robustly and migrate to the tumor bedwhere they recognize the infectious agent. At the same time, the secondTCR of these effector CD8 T cells recognize tumor antigens and executeeffector function, causing site-specific tumor regression andlong-lasting antitumor immunity. Complete and lasting tumor regressionwas seen in some treated animals. Overall, the present inventionharnesses the power of multiple arms of the immune system with promisingtranslational value, which can be used to target many types of solidtumors.

In one aspect, a purified population of dual-specific lymphocytes whichhave specificity for two or more antigens is provided, wherein apopulation of lymphocytes is isolated from a patient and wherein eachlymphocyte expresses an endogenous receptor for a tumor associatedantigen (TAA) and is genetically engineered to express an additionalexogenous receptor for a strong antigen. This population ofdual-specific lymphocytes can target and be activated by a plurality ofTAAs and the strong antigen. In a preferred aspect, the lymphocytes areCD8+ T cells.

In some aspects, the present invention is a composition comprising orconsisting essentially of a purified population of dual-specificlymphocytes and a pharmaceutically acceptable carrier.

In a further aspect, a method of producing an autologous population ofdual-specific lymphocytes specific for a plurality of tumor associatedantigens and at least one strong antigen is provided. The methodcomprising the steps of:

-   -   (a) isolating lymphocytes from a patient;    -   (b) purifying the tumor-specific lymphocytes from the isolated        lymphocytes; and    -   (c) genetically engineering the purified lymphocytes to express        a second receptor specific to at least one strong antigen,    -   wherein the resulting population comprises dual-specific        lymphocytes.

In some aspects, the method further comprises (d) culturally expandingthe dual-specific lymphocytes to increase the number of dual-specificlymphocytes.

In another aspect, a method of treating a patient with a tumor isprovided. The method comprises (a) administering to the patient aneffective amount of autologous dual-specific lymphocytes, and (b)injecting the patient with a strong antigen. In a preferred aspect, thestrong antigen is injected intratumorally.

In yet another aspect, a pharmaceutical composition is provided. Thepharmaceutical composition comprises or consists essentially of apopulation of autologous dual-specific lymphocytes reactive with aplurality of tumor associated antigens specific to a tumor andgenetically engineered to express a receptor reactive with a strongantigen; and a pharmaceutically acceptable carrier.

In another aspect, a kit for treating a tumor in a patient is provided,the kit comprising dual-specific lymphocytes autologous to the patientthat recognize a plurality of tumor associated antigens and a strongantigen, and a sufficient amount of strong antigen able to be injectedinto the patient.

In another aspect, a kit for producing a population of dual-specificlymphocytes autologous to a patient is provided. The kit includes (a) avector encoding a receptor specific to a strong antigen able to beexpressed in a cell.

The foregoing and other aspects and advantages of the invention willappear from the following description. In the description, reference ismade to the accompanying drawings which form a part hereof, and in whichthere is shown by way of illustration a preferred embodiment of theinvention. Such embodiment does not necessarily represent the full scopeof the invention, however, and reference is made therefore to the claimsand herein for interpreting the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-1D indicate ReACT shows significantly enhanced antitumorefficacy. FIG. 1A. The experimental scheme of ReACT (ReenergizedAdoptive Cell Transfer). Pmel-1 CD8 T cells are transduced in vitro toexpress a second TCR (OT-I) to generate T cells that could recognizeboth a TAA gp100 and a surrogate bacterial antigen OVA₂₅₇₋₂₆₄. Thesedual-specific CD8 T cells were expanded in vitro and transferred totumor-bearing mice followed by i.t. LM-OVA infection.

FIG. 1B. Dot plots show the intracellular IFNγ staining in Pmel-1⁺ orOT-I⁺ Pmel-1⁺ CD8 T cells after 6 hours of stimulation with gp100 or OVApeptide, respectively.

FIG. 1C. The B16-F10 tumor-bearing mice received the followingcombinations of treatments: mono-specific CD8 T cell transfer,mono-specific CD8 T cell transfer accompanied by i.t. injection ofLM-OVA, dual-specific CD8 T cell transfer or dual-specific CD8 T celltransfer accompanied by i.t. injection of LM-OVA. In each group, 5×10⁵CD8 T cells were transferred into each mouse. The individual tumorgrowth curves following each treatment as stated in (C) were analyzed byKruskai-Wallis with Dunn's multiple comparison tests. The number on topright represents the responder/total mice ratio.

FIG. 1D. The overall tumor growth is shown as mean volume ±s.e.m.

FIGS. 2A-2B shows the adjuvant effect of Listeria and the antitumoreffect of LM-OVA as a cancer vaccine. FIG. 2A. C57BL/6 mice withestablished B16-F10 tumors were treated with ACT of either OT-I cells(5×10⁵/mouse) alone or mixed Pmel-1 (2.5×10⁵/mouse) and OT-I cells(2.5×10⁵/mouse), followed by LM-OVA i.t. injection. The individual tumorgrowth curves are shown.

FIG. 2B. C57BL/6 mice were injected i.t. with either LM-OVA or LM-GP33one week after subcutaneous inoculation with B16-OVA tumor cells. Tumorgrowth in each group was monitored over time, and individual tumorgrowth curves are shown. In all experiments, mice that eradicated tumorswere defined as responders (shown in solid lines), while the remainingmice were defined as non-responders (shown in dashed lines). The numberon top right represents the responder/total mice ratio. Data shown arepooled from two to three independent experiments.

FIGS. 3A-3D show Polyclonal ReACT confers efficient tumor control andgenerates long-term protection. FIG. 3A. The schematic for generatingpolyclonal dual-specific CD8 T cells for ReACT therapy against solidtumors.

FIGS. 3A-3D show Polyclonal ReACT confers efficient tumor control andgenerates long-term protection. FIG. 3B. Four groups of tumor bearingmice received different treatment regimens including: polyclonalmono-specific CD8 T cell transfer (5×10⁵/mouse) with or without LM-OVAinfection and polyclonal dual-specific CD8 T cell transfer (5×10⁵/mouse)with or without LM-OVA infection. The responders (shown in solid lines)and non-responders (shown in dashed lines) in each were defined asdescribed in FIG. 1. The data were analyzed by Kruskai-Wallis withDunn's multiple comparison tests.

FIG. 3C. The individual growth curves of breast cancer E0771 tumorsafter receiving four different treatments as stated in (B) are shown.

FIG. 3D. Mice that eradicated their primary B16-F10 tumors werere-inoculated with 1×10⁴ B16-F10 cells (shown in left) and previouslyunencountered breast cancer cells (E0771, shown in right). As areference, B16-F10 tumor growth in naive mice is also shown. The numberof tumor free mice are shown, and were analyzed by Log-rank test. Datawere pooled from two independent experiments.

FIGS. 4A-4F show ReACT markedly increases tumor-specific CD8 T cellexpansion, function and tumor-targeted migration. C57BL/6 mice receivedvarious combinations of treatments described in FIG. 1. Ten days later,mice were euthanized to harvest tumor infiltrating immune cells for flowcytometric analysis. FIG. 4A. The representative plots are gated on CD8T cells and the numbers indicate the percentage of transferred (GFP+)cells.

FIG. 4B. The percentage and absolute number (normalized to tumor volume)of transferred CD8 T cells were calculated and shown in the plots.

FIG. 4C. Correlation plot shows the relationship between tumor sizes andfrequency of CD8 T cells within the tumor in each treatment group. Eachdata point represents an individual mouse.

FIG. 4D. The expression of CD44, KLRG-1, CXCR3 and granzyme B werecompared in dual-specific and mono-specific CD8 T cells by flowcytometry. Naïve CD8 T cells (CD44⁻) served as control.

FIG. 4E. The representative plots show the production of IFNγ and TNFαafter stimulation with gp100 peptide in vitro for 6 hours.

FIG. 4F. The percentage of IFNγ producing CD8⁺ T cells was calculatedand shown in the plot. Data shown are pooled from three independentexperiments.

FIGS. 5A-5J show ReACT alters the tumor immunosuppressivemicroenvironment and tumor-specific CD8 T cell phenotypes. FIG. 5A. Thefrequency of CD25⁺ Foxp3⁺ Tregs inside tumors from mice that receivedeach treatment described in FIG. 1 is shown in the dot plots.

FIG. 5B. The percentage and absolute number of Tregs normalized withtumor volume were calculated and plotted in the graphs. FIG. 5C.Correlation plot shows the relationship between tumor sizes andfrequency of Tregs inside the tumor.

FIG. 5D. The Teff/Treg ratios were calculated and plotted in the graphs.

FIG. 5E. The Teff/Treg ratios calculated and plotted in the graphs.

FIG. 5F. The frequency of CD11b⁺Gr-1⁺ MDSCs in tumors is shown in thedot plots.

FIG. 5G. The percentage and absolute number of CD11b⁺Gr-1⁺ MDSCsnormalized with tumor volume were enumerated and plotted in the graph.

FIG. 5H. The expression of iNOS was compared in CD11b⁺Gr-1⁺ cells fromuninfected and infected tumor-bearing mice and shown in histograms.

FIG. 5I. The CD11b⁺ cells were sorted from B16-F10 tumors treated witheither i.t. injection of LM-OVA or PBS. These cells were co-culturedwith activated CD8 T cells and the proliferation of T cells was assessedby ³H-thymidine incorporation and shown in bar graphs.

FIG. 5J. The expression of inhibitory receptors (LAG3, CTLA4, Tim3 andPD-1) was compared between dual-specific and mono-specific CD8 T cellsinside tumor and plotted in histograms. Data were pooled from threeindependent experiments.

FIGS. 6A-6B show the phenotypes of bone marrow derived dendritic cells(BMDCs) and polyclonal dual-specific CD8 T cells. FIG. 6A. Surfacemarker expression of BMDCs was analyzed by flow cytometry and shown inthe dot plot and histogram.

FIG. 6B. BMDCs pulsed with tumor cell lysates were used to generatepolyclonal dual-specific CD8 T cells. Histograms show the expression ofsurface phenotypic markers on the activated dual-specific CD8 T cells.

FIGS. 7A-7D show polyclonal ReACT increases tumor-specific CD8 T cellexpansion and function. Four groups of B16-F10 tumor bearing micereceived different treatment regimens including: polyclonalmono-specific CD8 T cell transfer (5×10⁵/mouse) with or without LM-OVAinfection and polyclonal dual-specific CD8 T cell transfer (5×10⁵/mouse)with or without LM-OVA infection. FIG. 7A. Dot plots show the frequencyof adoptively transferred CD8 T cells from each group in the peripheralblood ten days after treatments.

FIG. 7B. The frequency and number of adoptively transferred CD8 T cellsin the tumors were enumerated and shown in scatter plots.

FIG. 7C. The association plot shows the relationship between tumor sizesand the frequency of CD8 T cells in the tumors.

FIG. 7D. The expression of PD-1, KLRG-1 and granzyme B were comparedbetween dual-specific, mono-specific and naive CD8 T cells and shown inhistograms. Results are representative of 2 independent experiments,with n>3/group. * denotes P<0.05, ** denotes P<0.01, and *** denotesP<0.001.

FIGS. 8A-8E show polyclonal ReACT reduces Treg cells and increaseseffector/Treg ratios in the tumors. Tumor bearing mice received variouscombinations of therapy as described in Supplemental FIG. 2. FIG. 8A.The intratumoral Tregs were identified as CD25₊ Foxp3₊ and shown inrepresentative FACS pots gated on CD4₊ cells.

FIG. 8B. The frequency and number of Tregs were enumerated and shown inscatter plots.

FIG. 8C. The correlation plot shows the relationship between thefrequency of intratumoral Tregs and tumor sizes.

FIG. 8D. The CD8 Teff/Treg ratios and their association with tumor sizeswere calculated and shown in graphs. Results are representative of 2independent experiments, with n>3/group. ** denotes P<0.01.

FIG. 8E. The CD8 Teff/Treg ratios and their association with tumor sizeswere calculated and shown in graphs. Results are representative of 2independent experiments, with n>3/group. ** denotes P<0.01.

FIGS. 9A-9B show polyclonal ReACT reduces CD11b₊ cells in the tumors andalters their phenotype. Tumor bearing mice received various combinationsof therapy as described in Supplemental FIG. 2. FIG. 9A. The frequencyand absolute number of CD11b₊ myeloid cells from tumors were enumeratedand shown in scatter plots.

FIG. 9B. The iNOS expression in CD11b₊ cells isolated from tumor bearingmice with or without i.t. LM-OVA infection was analyzed by flowcytometry at day 3 p.i. and shown in the histogram. Results arerepresentative of 3 independent experiments, with n>3/group. * denotesP<0.05, ** denotes P<0.01, and *** denotes P<0.001.

DETAILED DESCRIPTION OF THE INVENTION

The present invention has been described in terms of one or morepreferred embodiments, and it should be appreciated that manyequivalents, alternatives, variations, and modifications, aside fromthose expressly stated, are possible and within the scope of theinvention.

The present disclosure provides a purified population of autologousdual-specific lymphocytes, methods of making and methods of using thesedual-specific lymphocytes to treat tumors in a patient.

Adoptive cell transfer (ACT) based immunotherapy in fighting cancer hasrecently attracted great interest. This approach includes engineeringand expanding the tumor-infiltrating lymphocytes, which can recognizetumor associated antigens (TAAs), followed by infusing them intopatients to induce a tumor specific immune response. Despite the recentsuccess in treating hematopoietic malignancies (Maus, et al 2014),limited efficacy has been achieved in treating other solid tumor(Restifo, et al 2012).

One of the main stumbling blocks in the use of ACT is the number oftransferred engineered T cells required to achieve therapeutic responseis too few once injected into the patient and TILs isolated frommalignant lesions readily lose their proliferative potential after exvivo expansion with a high dose of IL-2 (Kalia et al 2010; Pipkin et al2010).

To overcome this problem, autologous T cells are genetically engineeredT cell receptor (TCR) or chimeric antigen receptor (CAR) to equip themwith tumor reactivity, which has resulted in remarkable responses inhematological malignancies (Maude et al 2014). However, theheterogeneity of tumor cells makes this monoclonal T cell approach lessefficient to establish durable and complete regression of most tumors.Furthermore, even high numbers of fully activated tumor-specificcytotoxic CD8+ T cells can fail to induce tumor regression due to theirinsufficient recruitment to tumor tissue (Ganss and Hanahan 1998, Gansset al 2002 and Garbi et al 2004). The majority of solid tumors arestromal rich with disorganized vasculature, which creates physicalbarriers for efficient trafficking of therapeutic T cells to the tumorbed (Barnas, et al. 2010; Bellone et al 2013).

Lastly, another equally important major hurdle is the accumulation ofimmunosuppressive regulatory T cells (Tregs) and myeloid-derivedsuppressor cells (MDSCs) within the tumor microenvironment (Curiel et al2007 and Lu et al 2011), which usually leads to the progressive loss ofT cell effector function. Recent studies have shown that depletion ofTreg by using either cyclophosphamide (Le et al 2012) or CD25 Abs(Poehlein et al 2009), or MDSC removal by sunitinib (Ko et al 2009)restored tumor-specific T cell responses. With all these limitations,treating solid tumors such as melanoma with immunotherapy remainsdifficult.

The present disclosure provides a new strategy, using a population ofdual-specific lymphocytes that have polyclonal specificity for tumorassociated antigens and also specificity for a strong antigen incombination with immunization to the strong antigen intratumorally totarget and treat solid tumors. This method we have termed ReenergizedACT (ReACT). This method uses a strong antigen (e.g. a pathogen) notonly to break the immunosuppression, but also to drive the expansion andmigration of tumor-specific T cells to the very site of tumor. With thiscombinatorial approach, we have demonstrated that ReACT enhancesantitumor efficacy in comparison to either ACT or pathogen-based cancervaccine alone in primary tumor eradication and offers long-termprotection against reoccurrence in preclinical cancer models. Asdemonstrated in the examples, the current method not only reduces tumorburden but has resulted in complete regression of the tumor aftertreatment.

In some embodiments, the present disclosure provides a purifiedpopulation of dual-specific lymphocytes which have specificity for twoor more antigens. The term “dual-specific lymphocytes” refers tolymphocytes that have specificity for at least two different antigens.The dual specific lymphocytes therefore have at least two receptors thebind to two different antigens. In some embodiments, the dual-specificlymphocytes have specificity for at least one tumor associated antigenand at least one strong antigen. In some embodiments, the dual-specificlymphocytes may be engineered to have a second receptor for a secondtumor antigen and/or a second strong antigen. For example, adual-specific lymphocyte may have specificity for one TAA and one strongantigen, alternatively may have specificity for at least two TAA (e.g.one endogenous TAA and one exogenous TAA genetically engineered into thecell) and at least one strong antigen, alternatively may havespecificity for at least one TAA and at least two strong antigens, andthe like. Other combinations are also contemplated of at least one TAAand at least one strong antigen.

The present disclosure provides a method of producing an autologouspopulation of dual-specific lymphocytes that can target a plurality oftumor associated antigens and at least one strong antigen. This methodcomprises the steps of: (a) isolating lymphocytes from a patient; (b)purifying the tumor-specific lymphocytes from the isolated lymphocytes;(c) genetically engineering the purified lymphocytes to express a secondreceptor specific to a strong antigen, wherein the resulting populationcomprises dual-specific lymphocytes

The autologous dual-specific lymphocytes produced by the methods eachexpress a receptor for a tumor associated antigen (TAA) and aregenetically engineered to express an additional receptor for a strongantigen, wherein the population of dual-specific lymphocytes target aplurality of TAAs and the strong antigen.

The term “autologous” herein refers to lymphocytes that are obtainedfrom the patient to be treated. The term “lymphocyte” herein refers towhite blood cells that elicit a cell-mediated immune response. Suitablelymphocytes that may be used include, but are not limited to, CD8+ Tcells, CD4+ T cells, natural killer (NK) cells and combinations thereof.In a preferred embodiment, the lymphocytes are CD8+ T cells.

In some embodiments, the lymphocytes are tumor infiltrating lymphocytes(TILs). TILs are white blood cells that have left the bloodstream andmigrated into a tumor within a patient. TILs can be a mix of differenttypes of cells (i.e., T cells, B cells, NK cells, macrophages) invariable proportions, although T cells are normally the most prevalent.

Methods of isolating lymphocytes from a patient are known in the art.Most preferably, the lymphocytes are isolated from tumor tissue excisedfrom the patient. To create the population of cells to use for thegenetic manipulation one would harvest the tumor or a portion of thetumor from a patient as a source of lymphocytes or TILs. For example,one can plate fragments of the tumor in cell culture with cytokines tostimulate lymphocyte growth and expansion.

In some embodiments, a layer of irradiated feeder lymphocytes is used tosupport the culture of TILs but other methods such as the addition ofconditioned media or support cocktails could be employed. One suchexample of cytokine conditions used to stimulate lymphocyte growth isthe addition of 100 IU/ml of interleukin-2, but other cytokines, growthfactors and concentrations can be empirically determined and known byone skilled in the art such as the use of interleukin-7 orinterleukin-15. Cultures with strong growth will kill the tumor cellsleading to cultures enriched for lymphocytes. Next, the clones oflymphocytes are expanded and tested specifically for their ability tokill the primary tumor in a coculture system. Those clones which wereable to kill the tumor are selected for gene editing to create adual-specific lymphocytes. These dual-specific lymphocytes can targetnot just the tumor but also the strong antigen.

From the excised tumor or tumor fragment, a heterogeneous population oflymphocytes are isolated from the patient. By heterogeneous, thelymphocyte population includes more than one lymphocyte which isspecific for more than one tumor associate antigen (TAA). In otherwords, a population of lymphocytes (e.g. CD8+ T cells) is isolated inwhich the population contains more than one lymphocyte expressing TCRsthat can recognize more than one different TAA. In some embodiments, thedual-specific lymphocyte recognizes at least 2 to tens of thousands ofTAAs. Not to be bound by any theory, but since the T cells beingactivated are polyclonal, they can react to many different tumorassociated antigens that are specific to the tumor from which thelymphocytes are isolated. One advantage, among others, of this method isthat since the lymphocytes are isolated from the tumor of the patient,they contain a number of endogenous lymphocytes that recognize differentTAAs that are specifically expressed in the tumor of that specificpatient. These lymphocytes therefore are tumor-specific, patientspecific heterologous population providing an advantage over cells thatare engineered to only express a single TAA. These lymphocytes aregenetically engineered to express a second receptor for a strongantigen.

The second receptor that recognizes a strong antigen may be a second Tcell receptor (TCR) or chimeric antigen receptor (CAR). The method ofexpressing a second receptor in a lymphocyte can be done by standardmethods known in the art (e.g. transfection or transduction). Forexample, a receptor gene may be transduced into the lymphocyte usingmethods known in the art, for example, a retroviral vector for murinetransduction or a lentiviral vector for human transduction. The TCR orCAR is provided as a recombinant DNA molecule comprising all or part ofthe T-cell receptor nucleic acid sequence within a vector.

A CAR is transmembrane protein containing an extracellular portioncontaining a recognition or binding site for the strong antigen and atransmembrane and intracellular domain capable of signal transduction toactivate the lymphocyte (e.g. T cell). CAR are known in the art and canbe made using standard techniques. In one example, the chimeric receptoris a T-cell receptor or fragment thereof which recognize the strongantigen and activates the lymphocyte once bound to the strong antigen.For example, a suitable chimeric receptor is a chimeric containing thesingle chain variable region from a monoclonal antibody joined to the Fcreceptor section capable of mediating T-cell receptor signaltransduction. Another chimeric receptor comprises the antibody variableregion joined to the cytoplasmic region of CD28 from a T cell or asimilar region such as 41BB which can provide a T cell withco-stimulation signal necessary to activate the T cell. One can link thestrong antigen receptor of a CAR to an internal signal amplifier such asCD28 or 41BB as would be known by one of skill in the art. Suitablemethods of making genetically engineered antigen receptors, and chimericantigen receptors are known in the art, including, for example, asdetailed in Monjezi et al. Enhanced CAR T-cell engineering usingnon-viral Sleeping Beauty transposition from minicircle vectors.Leukemia. 2016 Aug. 5. doi: 10.1038/leu.2016.180; Mock et al. Automatedmanufacturing of chimeric antigen receptor T cells for adoptiveimmunotherapy using CliniMACS prodigy. Cytotherapy. 2016 August;18(8):1002-11. doi: 10.1016/j.jcyt.2016.05.009; Jin et al. Safeengineering of CAR T cells for adoptive cell therapy of cancer usinglong-term episomal gene transfer. EMBO Mol Med. 2016 Jul. 1;8(7):702-11. doi: 10.15252/emmm.201505869; An et al. Construction of anew anti-CD19 chimeric antigen receptor and the anti-leukemia functionstudy of the transduced T cells. Oncotarget. 2016 Mar. 1; 7(9):10638-49.doi: 10.18632/oncotarget.7079; Ren et al. Modification ofcytokine-induced killer cells with chimeric antigen receptors (CARs)enhances antitumor immunity to epidermal growth factor receptor(EGFR)-positive malignancies. Cancer Immunol Immunother. 2015 December;64(12):1517-29. doi: 10.1007/s00262-015-1757-6; Oldham R A, Berinstein EM, and Medin J A. Lentiviral vectors in cancer immunotherapy.Immunotherapy. 2015; 7(3): 271-84. doi: 10.2217/imt.14.108; Review;Urbanska et al. Targeted cancer immunotherapy via combination ofdesigner bispecific antibody and novel gene-engineered T cells. J TranslMed. 2014 Dec. 13; 12:347. doi: 10.1186/s12967-014-0347-2, the contentsof which are incorporated by reference in their entireties. A chimericantigen receptor is expressed by a chimeric receptor gene which can beplaced into a vector able to express the chimeric receptor gene.Suitable examples for the production of chimeric T-cell receptors andtheir corresponding genes from production can be found for example in,U.S. Pat. No. 5,830,755 and U.S. application Ser. No. 08/547,263, bothof which are incorporated by reference in their entireties.

In some embodiments, the genetically engineering of the lymphocytecomprises transducing the lymphocyte with a gene encoding for thereceptor to a strong antigen. By “genetic engineering” we mean thedesign and introduction of exogenous or foreign DNA into the lymphocyteby those methods known in the art. The term “transduction” means theintroduction of exogenous or foreign DNA into the lymphocyte. Suitablemethods of transducing the lymphocytes are known in the art and include,but are not limited to, retroviral transduction, adenoviral or otherviral transduction methods, electroporation, transfection usinglipofection, calcium phosphate, gene transfer or other procedures knownto one skilled in the art, including, for example, as discussed inSambrook et al. (1989), “Molecular Cloning. A Laboratory Manual,” ColdSpring Harbor Press, Plainview, N.Y., which is incorporated by referencein its entirety. Suitable vectors and kits are commercially availableand known to one skilled in the art for expressing the second receptor.Further, genetic engineering also includes any method which employs anynumber of enzyme systems that one could use to perform gene editing onthe receptor and/or vector, and include, but are not limited to,CRISPR/Cas (Clustered regularly interspaced short palindrome repeats(CRISPRs)), CRISPR-associated Zinc-finger nucleases (ZFNs), andtranscription-activator-like effector nucleases (TALENs). These arechimeric nucleases composed of programmable, sequence-specificDNA-binding modules linked to a nonspecific DNA cleavage domain. Methodsof genetically engineering a cell to express a second receptor are knownin the art.

Suitable vectors are known in the art and include expression vectorsthat comprise at least one expression control element operably linked tothe nucleic acid sequence. The expression control elements are insertedin the vector to control and regulate the expression of the nucleic acidsequences and are known in the art, for example promoters and/orenhancers. Suitable vectors include, but are not limited to, adenovirus,retrovirus, cytomegalovirus (CMV), MMLV, SV40, and the like. Additionalpreferred operational elements include, but are not limited to, leadersequences, termination codons, polyadenylation signals and any othersequences necessary for the appropriate transcription and expression ofthe nucleic acid. Suitable expression systems and expression vectors areknown in the art and one skilled in the art is able to select anexpression vector suitable for the cell chosen. It is understood thatthe vectors may contain additional elements beneficial for properprotein expression, and are well known in the art. In some embodiments,the vectors will include a selectable marker, e.g. ampicillin resistanceand/or fluorescent protein expression (e.g. GFP/RFP), that allows forselection of the transduced and/or transformed cells.

In a non-limiting example, the vector to be used for transfection toexpress a receptor to a strong antigen could be produced as follows:expand peptide-specific T cells isolated from PBMCs of a healthy donorby incubating them with the pathogenic peptide in vitro. For example,for BCG, Geluk et al. identified two HLA-A*0201 restricted CD8 T cellepitopes of Ag85B (BCG derived antigen), these two peptides spanned aa145-152 FIY AGSLS (SEQ ID NO:1) and aa 199-207 KLV ANNTRL (SEQ ID NO:2).In this example, one would choose one of the two peptides to expand Tcells in vitro. Next, one can isolate peptide-specific T cells byfluorescence-activated cell sorting (FACS), selecting for CD8 T cellscarrying the activation marker CD45RO (marker for effector and memory Tcells). DNA isolated from these T cells can be subjected to nextgeneration sequencing to identify the most abundant variable regions ofalpha and beta chain of clonal T cell receptors (TCRs). The sequencingresults will indicate the dominant TCR. The dominant TCR sequencesidentified will be used to construct a vector that co-expresses bothalpha and beta chains of the TCR, which can then be used to express theTCR in lymphocytes.

In a preferred embodiment, the lymphocytes are transfected using aretroviral or lentiviral vector system, wherein the lymphocytes areincubated with retroviral or lentiviral supernatant in a concentrationof about 1×10² to about 1×10¹⁰ viral particles per ml. Preferably,lentiviral vectors are used in human subjects.

In some embodiments, the vector further comprises a selection markerthat can be used to select the transduced cells.

The term “strong antigen” refers to an antigen able to induceproliferation of lymphocytes (including T cells). The strong antigen isable to induce proliferation when exposed to the lymphocytes either invitro or in vivo. The term strong antigen may refer to a single antigenor a set of antigens. Suitable strong antigens include, but are notlimited to, pathogens (e.g. viral antigens or bacterial antigens) andalloantigens. In some embodiments, the pathogen may be an attenuatedform of the microorganism, any pathogen that is suitable for vaccinationand clinical use in humans. Suitable strong antigens include, but arenot limited to, live or attenuated bacteria, virus, human leukocyteantigen, major histocompatibility complex, a pathogenic protein or aprotein or peptide fragment of any of the aforementioned.

Alloantigens are antigens derived from genetically non-identical membersof the same species. Alloantigens may be tissues, cells, proteins, orpeptides, e.g. HLAs.

In some embodiments, the strong antigen is an antigen from a pathogen,wherein the pathogen is selected from the group consisting of listeriamonocytogenes, Bacillus Calmette-Guérin, tetanus, diphtheria,adenovirus, herpes simplex virus, vaccinia virus, myxoma virus,poliovirus, vesicular stamatis virus, measles virus, Newcastle diseasevirus and combinations thereof. Other suitable pathogens that are inhuman trials and may be used in the practice of the present technologyinclude:

-   -   (a) Adenovirus 5 (dl1520 derivative) Squamous cell carcinoma of        head and neck (approved drug in China; intratumoural);    -   (b) Adenovirus 5 (PSE-E1A and E3 deleted) Prostate (I;        prostatic);    -   (c) Herpes simplex virus 1 (ICP34.5 defective) Glioblastoma        multiforme (II; intratumoural);    -   (d) Vaccinia virus (thymidine kinase knockout and expressing        granulocyte-macrophage colony-stimulating factor) Advanced liver        tumours (I-II; intratumoural);    -   (e) Reovirus (reolysin) Superficial tumours (I; intralesional);    -   (f) Newcastle disease virus (PV701) Bladder, squamous cell        carcinoma of head and neck and ovarian (I-II; intravenous);    -   (g) Measles virus (V protein knockout and expressing the        reporter carcinoembryonic antigen or the effector sodium iodide        symporter) Ovarian (I; intratumoural), glioma.

In some cases, a pathogen marker can be used in order to track, find orboost responsiveness to the pathogen. In the example shown, a portion ofthe ovalbumin protein was expressed as an antigen secondary to thepathogen. Specifically, OVA SIINFEKL peptide (SEQ ID NO:3) (respondingto recombinant Listeria Monocytogenes-OVA, LM-OVA).

The methods of producing dual-specific lymphocytes of the presenttechnology include the step of culturing the lymphocytes in vitro.During this culturing step, the lymphocytes are selected/activated andpurified that are specific to one or more tumor associated antigens bymethods known in the art. For example, one method to culture and purifythe lymphocytes that are specific to tumor cells of the patient is toculture the lymphocytes in the presence of tumor cells isolated from thepatient or to one or more tumor associated antigens. In the preferredembodiment, the lymphocytes are cultured with tissue, cells, or parts oftissue from the tumor of the patient. Otherwise, suitable tumorassociated antigens to culture the cells with are known in the art andmay be specific to the specific type of tumor being treated. Forexample, a listing of tumor antigens that can be used for culturing canbe found on-line in the Peptide Database, for example, but not limitedto, the TAAs such as AFP (Alpha-feto protein), ALK gene rearrangements,B-cell immunoglobulin gene rearrangement, B2M (Beta-2 microglobulin),BCR-ABL, CA 15-3 (Cancer antigen 15-3), CA 19-9 (Cancer antigen 19-9),CA-125 (Cancer antigen 125), Calcitonin, CEA (Carcino-embryonicantigen), Chromogranin A (CgA), DCP (Des-gamma-carboxy prothrombin),EGFR mutation, Estrogen and Progesterone receptors, Fibrin/Fibrinogen,Gastrin, hCG (Human chorionic gonadotropin, also called Beta-hCG),HER2/neu, JAK2 mutation, KRAS mutation, LD (Lactate dehydrogenase),Monoclonal immunoglobulins, PSA (Prostate specific antigen), SMRP(Soluble mesothelin-related peptides), T-cell receptor generearrangement, Thyroglobulin, 21-gene signature (Oncotype DX®) and70-gene signature (MammaPrint®), and others known in the art. A suitablemethod for culturing the lymphocytes can be found in van der Bruggen P,Stroobant V, Vigneron N, Vanden Eynde B. Peptide database: Tcell-defined tumor antigens. Cancer Immun. 2013, which is incorporatedby reference in its entirety.

The culturing step may also include a step of culturally expanding thetumor cells to increase the number of tumor specific lymphocytes thatare able to be transferred back into a patient. Suitable methods forexpanding the in vitro cultured lymphocytes are known in the art. In oneembodiment, the specific expanding step amplifies a subpopulation of Tcells whose endogenous T cells are directed to a plurality of tumorassociated antigens from the tumor of the patient. This produces aheterogeneous population of autologous lymphocytes (e.g. CD8+ T cells)that can recognize a plurality of tumor associated antigens specific tothe patient. These expanded heterogeneous population of lymphocytes arethen further transduced with the second receptor (e.g. an exogenous TCRor chimeric antigen receptor) to produce the dual-specific lymphocytes.

In some embodiments of the method, the genetically-engineereddual-specific lymphocytes are cultured in the presence of both the tumorassociated antigens (or tissue or cells from the tumor of the patient)and the strong antigen to increase the number of cells reactive to boththe TAA and the strong antigen. In other words, the dual-specificlymphocytes are stimulated with the strong antigen and/or a plurality ofTAAs in culture to proliferate. In some embodiments, the lymphocytes,for example CD8+ T cells are cultured with IL-2 to increase the numberof dual-specific lymphocytes in the culture.

In some embodiments, the lymphocytes are activated with the strongantigen to result in proliferation of the dual-specific lymphocytes. Insome embodiments, the lymphocytes are exposed to the strong antigen forgreater than one hour, in some embodiments for at least 24 hours. Thelymphocytes are preferably exposed to the strong antigen continuouslyfor this time. Suitably, pathogenic antigens are exposed for at least 1hour to the lymphocytes, including at least 2 hours, at least 3 hours,at least 4 hours, at least 5 hours, at least 6 hours, at least 10 hours,at least 12 hours, at least 14 hours, at least 16 hours, at least 18hours, at least 20 hours, at least 24 hours, and includes any amount oftime in between. Suitably, alloantigens are exposed to the lymphocytesfor at least 24 hours, alternatively at least 30 hours, alternatively atleast 48 hours. Suitable amounts of strong antigen to expose thedual-specific lymphocytes will depend on the strong antigen, and areable to be determined by one skilled in the art. For example, the strongantigen may be provided in a concentration of about 0.1 to about 1 mMfor proteins, peptides or cellular components. For infectious orinactivated viral particles, they can be provided at a ratio of from1-1000 viral particles per cell in culture. Similarly, the number ofallogenic cells that may be used can be from 1 to 100 per lymphocyte inculture. In some embodiments, the strong antigen is combined with one ormore TAAs, including, in some embodiments, the inclusion of tissue orcells from the tumor extracted from the patient.

As used herein, the term plurality means two or more.

As used herein the term patient and subject can be used interchangeably.The patient is suitably a mammal, more preferably a human. In someembodiments, the compositions and methods may be used to treat a mammal,for example, a human, a chimpanzee, a mouse, a rat, a dog, a cat, ahorse or other livestock. In the most preferably embodiment, the methodis used to treat a human.

The present disclosure provides methods of treating a patient with atumor. The method comprises administering to the patient an effectiveamount of the autologous dual-specific lymphocytes described herein orautologous population of dual-specific lymphocytes made by the methodsdescribed herein and injecting the patient with a strong antigen, andpreferably injecting the strong antigen intratumorally. Pathogens havenatural predilictions or tropisms for infection and replication incertain areas of the body. Influenza infects and damages the respiratorytract primarily but the central nervous system only secondarily or inrare cases. Poliovirus infects and damages the central nervous systemprimarily but the respiratory system only secondarily. Using theknowledge of a pathogens natural tropism, one of skill in the art couldselect a strong antigen from a pathogen that had tropism for thelocation of the tumor to be treated. For example, if one wanted to treata lung tumor, one might use as the strong antigen of choice a strongantigen from influenza and then use a strategy of injecting orintranasally delivering an influenza vaccine. Though the preferredmethod of local delivery in many cases will be injection, othermechanisms of delivering the strong antigen to the site of the tumorcould be used such as intranasal delivery, gavage, lavage, or topicaldelivery.

By “treating” or “treatment” we mean the management and care of asubject for the purpose of combating and reducing the tumor. Treatingincludes the administration of a dual-specific lymphocytes of thepresent invention to reduce, inhibit, ameliorate and/or improve theonset of the symptoms or complications, alleviating the symptoms orcomplications of the tumor, or eliminating the tumor. Specifically,treatment results in the reduction in tumor load or volume in thepatient, and in some instances, leads to regression and elimination ofthe tumor or tumor cells. As used herein, the term “treatment” is notnecessarily meant to imply cure or complete abolition of the tumor.Treatment may refer to the inhibiting or slowing of the progression ofthe tumor, reducing the incidence of tumor, reducing metastasis of thetumor, or preventing additional tumor growth. In some embodiments,treatment results in complete regression of the tumor.

By “ameliorate,” “amelioration,” “improvement” or the like we mean adetectable improvement or a detectable change consistent withimprovement occurs in a subject or in at least a minority of subjects,e.g., in at least about 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%,70%, 75%, 80%, 85%, 90%, 95%, 98%, 100% or in a range about between anytwo of these values. Such improvement or change may be observed intreated subjects as compared to subjects not treated with thedual-specific lymphocytes and immunization with a strong antigen of thepresent invention, where the untreated subjects have, or are subject todeveloping, the same or similar tumor.

In some embodiments, the injecting step (in other words, the in vivoimmunization step) of the strong antigen into the tumor serves toactivate the adoptively transferred dual-specific lymphocytes (e.g. CD8+T cells) and to further home or target the transferred lymphocytes tothe tumor. Not to be bound by any theory, it is believed that theinjection of the strong antigen to the tumor cells overcomes theimmunosuppression of the tumor environment and results in a largeramount of the transferred dual-specific lymphocytes (e.g. CD8+ T cells)being recruited to the tumor which leads to increase tumor killing andtumor cell death.

In some embodiments, the method comprises administering an effectiveamount of the compositions described herein comprising a population ofautologous dual-specific lymphocytes and a pharmaceutically acceptablecarrier and injecting/immunizing the patient with the strong antigen.

In the preferred embodiment, the compositions or dual-specificlymphocytes are administered intravenously into the patient. In someembodiments, other methods of administration are contemplated, forexample, intra-arterially, intratumorally, parenterally, and the like.

The injecting of the strong antigen may be administered co-currentlywith the administration (adoptive transfer) of the dual-specificlymphocytes or can be done sequentially. In some embodiments, a secondinjection of the strong antigen is performed into the tumor. In otherembodiments, multiple injections of the strong antigen or multipledeliveries of the genetically altered patient immune cells over manymonths or years could be beneficial. A physician would monitor riskprofiles and tumor burden to empirically determine a treatment schedulefor a given patient.

A pharmaceutical composition comprising: a population of autologousdual-specific lymphocytes reactive to at least one tumor associatedantigen and containing a genetically engineered receptor reactive with astrong antigen a pharmaceutically acceptable carrier. The dual-specificlymphocytes are selected to be reactive with a plurality of tumorassociated antigens specific to a tumor.

By “pharmaceutically acceptable carrier” we mean any and all solvents,dispersion media, antibacterial and antifungal agents, isotonic agents,solutions or media and the like that are physiologically compatible anddo not result in harm to the dual-specific lymphocytes duringpreparation, storage or administration. The pharmaceutical compositionsmay optionally include one or more additional ingredients depending onthe mode of administration and the dual-specific lymphocytes or strongantigen to be administered to maintain the activity of the dual-specificlymphocytes or strong antigen during storage, preparation andadministration. Suitably, in some embodiments, the pharmaceuticalcomposition may contain additives such as pH-adjusting additives,anti-microbial preservatives, stabilizers and the like. Preferably, thecarrier is suitable for intravenous, parenteral, intraperitoneal, orintramuscular administration. Pharmaceutically acceptable carriersinclude sterile aqueous solutions or dispersions, including for example,saline or phosphate buffer saline. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with thelymphocytes, use thereof in the pharmaceutical compositions of theinvention is contemplated. Additional agents or therapies can also beincorporated into the compositions.

The pharmaceutical compositions described herein may be formulated withthe lymphocytes in a pharmaceutical carrier in accordance with knowntechniques. See, e.g., Remington, The Science And Practice of Pharmacy9th Ed. (A. R. Gennaro, ed., Mack Publishing Co., Easton, Pa., 1995).Also, the strong antigens may be formulated with a pharmaceuticalcarrier in accordance with known techniques for injection.

The inventor has surprisingly discovered a method to boost T cellresponses to tumor by artificially engineering a T cell to respond to asecond strong antigen in addition to polyclonal recognition of tumorantigens, as described in the examples. In the following example, CD8 Tcells from the Pmel-1 CD8 transgenic mouse in which 95% of CD8 T cellsexpress a T cell receptor (TCR) namely V alpha 1 and V beta 13. These Tcells recognize an epitope of melanoma tumor associated antigen (TAA)gp100. Using a retroviral vector, OT-I TCR (V alpha 2 and V beta 5),which recognizes ovalbumin (OVA) residues 257-264 and produces greenfluorescent protein (GFP) was transduced into the Pmel-1 CD8 T cellsresulting in cells which would express three important substances whichinclude: receptors for the gp100 tumor associated antigen; receptors forOVA which can be inoculated into the tumor to additionally target the Tcells to the tumor environment and boost their responsiveness; and GFP amolecule useful in the isolation, purification and later tracking of theartificially engineered cells.

Other types of immune cells, tumor antigens, pathogens and pathogenmarkers may be used.

The population of dual-specific lymphocytes, compositions and methods ofthis disclosure can be used to treat a patient suffering from a tumor.More preferably, the tumor is a solid tumor. Suitable tumors that can betreated by the methods described herein include, but are not limited to,a tumor selected from the group consisting of a sarcoma, a carcinoma anda melanoma. Suitable solid tumors that can be treated by the methodinclude, but are not limited to, brain and other central nervous systemtumors; head and/or neck cancer; breast tumors; circulatory systemtumors (e.g. heart, mediastinum and pleura, and other intrathoracicorgans, vascular tumors and tumor-associated vascular tissue); excretorysystem tumors (e.g. kidney, renal pelvis, ureter, bladder, other andunspecified urinary organs); gastrointestinal tract tumors (e.g.esophagus, stomach, small intestine, colon, colorectal, rectosigmoidjunction, rectum, anus and anal canal), tumors involving the liver andintrahepatic bile ducts, gall bladder, other and unspecified parts ofbiliary tract, pancreas, other and digestive organs); oral cavity;uterine cancer, ovarian cancer, penile cancer, prostate cancer,testicular cancer; respiratory tract tumors (e.g. nasal cavity andmiddle ear, accessory sinuses, larynx, trachea, bronchus and lung, e.g.small cell lung cancer or non-small cell lung cancer); skeletal systemtumors (e.g. bone and articular cartilage of limbs, bone articularcartilage and other sites); skin tumors (e.g. malignant melanoma of theskin, non-melanoma skin cancer, basal cell carcinoma of skin, squamouscell carcinoma of skin, mesothelioma, Kaposi's sarcoma); and tumorsinvolving other tissues. In some preferred embodiments, which may becommercially relevant include, but are not limited to, bladder cancerusing BCG as strong antigen, pancreatic cancer using listeria as strongantigen, melanoma using vaccinia as strong antigen and lung cancer usinginfluenza as strong antigen.

This disclosure also provides kits. The kits can be suitable for use inthe methods described herein. Suitable kits include a kit for treating atumor comprising genetically engineered autologous dual-specificlymphocytes described above and an injectable composition comprising thestrong antigen.

In some embodiments, a kit for producing dual-specific lymphocytes arecontemplated. In one embodiment, the kit comprises a vector encoding areceptor specific to a strong antigen able to be expressed in a cell isprovided.

In another embodiment, a kit comprising: (a) a vector encoding areceptor specific to a strong antigen able to be expressed in a cell;(b) a system for transducing the vector into lymphocytes; and (c)instructions for isolating tumor infiltrating lymphocytes from a tumorof a patient and transducing them with the strong antigen receptor areprovided. The cell is preferably a lymphocyte, more preferably a CD8+ Tcell.

In some embodiments, kits for carrying out the methods of the presentdisclosure are provided. For example, a kit for producing a populationof dual-specific lymphocytes, more specifically CD8+ T cells isprovided. The kit provides a vector encoding a receptor specific to astrong antigen and the strong antigen. Instructions are provided thatdescribes the method of isolated tumor-specific lymphocytes from apatient, culturing the isolated lymphocytes from the patient,transducing the lymphocytes to express the receptor for the strongantigen, and methods of using the duel specific lymphocytes fortreatment of a tumor are provided. In some embodiments, the instructionsfurther provide methods for culturally expanding and/or activating thelymphocytes.

The following non-limiting examples are included for purposes ofillustration only, and are not intended to limit the scope of the rangeof techniques and protocols in which the compositions and methods of thepresent invention may find utility, as will be appreciated by one ofskill in the art and can be readily implemented.

Example 1: Population of Dual-Specific CD8T Cells which Recognize aPlurality of Tumor Associated Antigen Specific for the Subject and aBacterial Antigen and Use for Treating of Solid Tumors

Immunosuppressive tumor microenvironment, insufficient migration andreduced effector function of tumor-specific T cells are the main hurdlesthat hamper the efficacy of immunotherapy in treating solid tumors. Inthis example, we combined the strength of adoptive cell transfer (ACT)and pathogen-based cancer vaccine and developed an innovative strategy,Reenergized ACT (ReACT), to treat solid tumors. ReACT uses a pathogennot only to break the immunosuppression, but also to drive the expansionand migration of tumor-specific T cells to the very site of tumor. Withthis combinatorial approach, we have demonstrated that ReACT enhancesantitumor efficacy in comparison to either ACT or pathogen-based cancervaccine alone in primary tumor eradication; and offers long-termprotection against reoccurrence in preclinical caner models.

To overcome the hurdles of immunosuppression and induce a vigorousantitumor T cell response, we combined the strength of ACT andpathogen-based cancer vaccines with a new strategy named Reenergized ACT(ReACT). To bridge ACT with a pathogen, we genetically engineeredpopulations of tumor-reactive CD8 T cells with a second T cell receptor(TCR) specific to a bacterial antigen to create dual-specific CD8 Tcells (i.e., a single T cell capable of recognizing two antigens). Thistechnology is based on a system developed by Kershaw and colleagues(12,13). However, our dual-specific CD8 T cells have advantages anddiffer from Kershaw in that we have a population of CD8 T cells whichexpress a plurality of different TCR specific to different tumorantigens that additionally express a genetically engineered TCR specificto a strong antigen, in this Example a TCR specific for the bacteriaListeria. We then used a pathogen-based vaccine to drive the robustexpansion of adoptively transferred bacteria- and tumor-(dual) specificT cells, recruit them to the tumor site, and concomitantly reverseimmunosuppression in the tumor microenvironment. This combined approachhas demonstrated robust efficacy in primary tumor eradication andlong-term protection against recurrence in preclinical cancer models.Not to be bound by any theories, but it is our belief that ourengineered dual-specific CD8 T cells provides a superior immune responseto the tumor as opposed to ACT using dual-specific CD8 T cells that aregenetically engineered to express a specific tumor antigen which may notreact to all tumor cells, leaving some tumor cells that do not expressthat specific tumor antigen or express low levels of the specific tumorantigen to expand and replace the targeted tumor cells.

Results

ReACT Enhances Antitumor Efficacy

First, we used a well-established mouse B16-F10 melanoma model (14) totest the antitumor efficacy of ReACT. To generate dual-specific CD8 Tcells, we started with Pmel-1 CD8 T cells, which express a TCR (Vα1 andVβ13) that recognizes the gp100 epitope of murine melanoma (14). Thesecells were then genetically engineered to express OT-I TCR (Vα2 and Vβ5)by retroviral transduction in vitro (FIG. 1A). OT-I recognizes ovalbumin(OVA) residues 257-264, which served as a surrogate bacterial antigenexpressed in a recombinant LM-OVA. We chose Listeria as a model organismbecause it is amenable to clinical use, and attenuated Listeria, likemany other pathogen-based cancer vaccines, has shown promising antitumoreffects in multiple cancer models in humans (ClinicalTrails.gov) andmice (9). To validate dual-specificity, control (empty vectortransduced; referred to as mono-specific CD8 T cells henceforth) andOT-I-TCR transduced (referred to as dual-specific CD8 T cellshenceforth) Pmel-1 cells were stimulated by antigenic peptides.Mono-specific CD8 T cells produced IFNγ after stimulation with gp100 butnot OVA₂₅₇₋₂₆₄ peptide (FIG. 1B). In contrast, dual-specific CD8 T cellsresponded to both gp100 and OVA₂₅₇₋₂₆₄ peptides (FIG. 1B).

To test the ability of transduced mono-specific or dual-specific CD8 Tcells to control melanoma in a therapeutic setting, a small number ofcells (5×10⁵/mouse) were adoptively transferred into C57BL/6 mice withestablished subcutaneous B16-F10 melanoma tumors. Consistent withpreviously published data (14), both ACT regimens failed to prevent thetumor growth (FIG. 1C). However, when dual-specific CD8 T cells wereadministered in combination with a low dose of LM-OVA (ReACT), there wassignificant tumor regression in all mice and the majority of mice (7 outof 10) had complete eradication (FIGS. 1C-1D). Notably, antitumoreffects required that mice were treated with both dual-specific T cellsand LM-OVA as tumor growth was only slightly and transiently suppressedin mice that received mono-specific CD8 T cells and LM-OVA (FIGS.1C-1D). Together, these results validate the feasibility of our approachand clearly show that ReACT leads to significantly enhanced antitumorefficacy.

The Adjuvant Effect of Listeria

It is possible that ReACT mediated tumor eradication was due to abystander anti-bacterial effect from the dual-specific CD8 T cells. Totest this possibility, we first transferred OT-I (OVA/bacteria-specific,non-transduced) CD8 T cells either alone or with Pmel-1 (tumor-specific)CD8 T cells into B16-F10 melanoma tumor bearing mice, and thenintratumorally administrated LM-OVA. Regardless of the robust expansionof OT-I cells in response to LM-OVA infection and their migration totumors, no obvious therapeutic benefit was seen in the OT-I+LM-OVA groupas compared to FIG. 1D (FIG. 2A and data not shown). In the same vein,bystander OT-I response to LM-OVA only conferred transient adjuvanteffects and failed to eradicate tumors even in the presence of Pmel-1cells (FIG. 2A and data not shown). These results together with the datashown in FIG. 1 demonstrated that LM-OVA infection either withmono-specific T cells alone (Pmel-1 or OT-I) or mixed mono-specific Tcells (Pmel-1 and OT-I) was insufficient to eradicate tumors. Withoutexpansion of adoptively transferred tumor-specific CD8 T cells, LM-OVAshows limited adjuvant effects in tumor control.

The Antitumor Effect of LM-OVA as a Cancer Vaccine

Recombinant Listeria expressing TAAs can serve as cancer vaccines totreat solid tumors (9). To test if a LM-based vaccine could confersimilar tumor control as seen by ReACT, we compared two recombinantstains of Listeria, LM-OVA (expressing LCMV glycoprotein 33-41 residues)and LM-GP33 (irrelevant control GP33 peptide) in the B16-OVA melanomatumor model. To test proof-of-principle and for simplicity, we usedOVA₂₅₇₋₂₆₄ as a surrogate tumor antigen as reported previously (15). Weadministrated LM-OVA and LM-GP33 i.t. to C57BL/6 mice with establishedB16-OVA melanoma and followed the tumor progression over time.Consistent with previous published work (16), LM-OVA led to a greatertumor control and 25% eradication when compared with LM-GP33 treatedmice (FIG. 2B). Nonetheless, this approach did not render robust tumoreradication as seen in ReACT treated mice (FIG. 1D). Taken together, ourdata suggest that combinatorial treatment with ACT and a pathogen-basedcancer vaccine leads to much greater tumor control than either treatmentalone.

Polyclonal ReACT Eradicates Tumor and Generates Long-Term Protection

Given the lack of well-defined TAAs for most human tumors, and theadvantages of using naturally occurring tumor-infiltrating lymphocytes(TILs) that recognize multiple TAAs to treat cancer patients (2), wefurther tested proof-of-principle by generating polyclonal CD8 T cellsthat target one bacterial antigen and multiple tumor antigens (FIG. 3A).For simplicity, we used B16-F10 cell lysate pulsed DCs to stimulatenaïve CD8 T cells to differentiate them into effector cytotoxic T cells(CTLs) that recognize various B16-F10 derived tumor antigens as shownpreviously (17). These cells were then genetically engineered to expressthe OT-I TCR and are referred to as polyclonal dual-specific CD8 T cells(FIG. 3A and FIGS. 6A-6B).

In line with the preceding observations, transfer of neithermono-specific nor dual-specific polyclonal CD8 T cells alone generatedtherapeutic responses against tumor growth in the absence of LM-OVAinfection (FIG. 3B). The combination of polyclonal mono-specific CD8 Tcells with LM-OVA infection only resulted in tumor elimination in 1 of11 mice (FIG. 3B). Strikingly, combined polyclonal dual-specific CD8 Tcells and LM-OVA infection (ReACT) led to complete tumor eradication inthe majority of mice (11 of 16) (FIG. 3B). Similar results were obtainedin the E0771 breast cancer model (FIG. 3C), demonstrating that thistherapy could potentially be applied to various types of solid tumors.

To test if this combined therapy could generate immunological memorythat protects the hosts from tumor recurrence, we challenged mice thathad eradicated primary melanoma (B16-F10) tumors with a lower dose ofB16-F10 cells on the left flank, and with a previously unencounteredcancer line (E0771 breast cancer cells) on the right flank. The majorityof these mice (7 out of 10) were resistant to B16-F10, whereas nonerejected the E0771 (FIG. 3D). As expected, naïve mice did not rejecteither B16-F10 or E0771 tumors (FIG. 3D). These data illustrate that thepolyclonal ReACT approach not only provides an enhanced immune responseto eradicate primary tumor, but also establishes long-term protectiveimmunity that prevents tumor relapse.

ReACT Increases CD8 T Cell Expansion, Function and Tumor-TargetedMigration

The remarkable antitumor effect of this combined strategy prompted us tostudy the tumor-specific CD8 T cell responses. Without preconditioningor additional adjuvants, very low frequencies and numbers of transferredCD8 T cells were detected in the tumors from mice that only receivedmono- or dual-specific CD8 T cell transfer alone, as reported previously(14) (FIGS. 4A-4B and FIGS. 7A-7B). This is not surprising given thatthe number of transferred cells was low and in vivo expansion followingACT was lacking. Interestingly, the intratumoral LM-OVA infectionslightly increased the mono-specific CD8 T cell infiltrating tumors,which is likely in response to the chemotactic inflammation. Morestrikingly, a significant amount of transferred CD8 T cells weredetected in tumors of mice that received bacterial infection combinedwith dual-specific CD8 T cell adoptive transfer (FIGS. 4A-4B and FIGS.7A-7B). Importantly, frequencies of CD8 T cells recruited to tumorsinversely correlated with tumor size in all treatment groups (FIG. 4Cand FIG. 7C). Furthermore, the dual-specific CD8 T cells displayed anactivated phenotype (CD44^(hi), KLRG-1^(hi) and granzyme B^(hi)) (FIG.4D and FIG. 7D), accompanied by high expression of the chemokinereceptor CXCR3, which has been shown to contribute to improved T cellmigration to tumors (18). More strikingly, we observed a significantnumber of multi-potent CD8 T cells producing both IFNγ and TNFα in onlymice receiving the combined treatment (FIGS. 3E-3F). Together, theseresults suggest that the dual-specific CD8 T cells in response tobacterial infection robustly expand, acquire effector function andmigrate to the site of tumor, which in turn results in enhanced tumorcontrol.

ReACT Reverses the Immunosuppressive TME and Recruits CD8 T Cells to theTumor

To assess whether our approach could alter the TME to synergisticallyimprove the tumor-specific CD8 T cell response, we examined two majorimmunosuppressive cells inside the tumor, Tregs and myeloid derivedsuppressive cells (MDSCs). The intratumoral LM-OVA infectionsignificantly reduced the frequency of CD4⁺ CD25⁺ Foxp3⁺ Tregsregardless of the type of CD8 T cells transferred (mono- ordual-specific) (FIGS. 5A-5B and FIGS. 8A-8B). Notably, the frequency ofTregs in all treated mice positively correlated with tumor size (FIG. 5Cand FIG. 8C). Interestingly, the effector/Treg ratio only increased inmice that received dual-specific CD8 T cells (FIG. 5D and FIG. 8D),owing to the robust expansion of effector cells as shown in FIGS. 5A-5Band FIGS. 7A-7B. Furthermore, the effector/Treg ratio inverselycorrelated with tumor size (FIG. 5E and FIG. 8E). Together, this showsthat the ratio between effector CD8 T cells and Tregs is a criticalfactor that determines the final outcomes of different treatments.

Another important type of suppressive cell, CD11b⁺Gr1⁺ MDSCs, were alsosignificantly reduced by LM-OVA infection (FIGS. 5F-5G and FIG. 9A).This is consistent with previous findings that Listeria can directlyinfect MDSCs (19), which likely makes them susceptible to cytotoxic Tcell mediated killing. Furthermore, Listeria infection can convert MDSCsinto immune stimulatory cells (19,20). By the same token, we observedthat the intratumoral Listeria infection caused elevated iNOS expressionin CD11b⁺Gr1⁺ cells (FIG. 5F and FIG. 9B). To further test if thisphenotypic change correlated with decreased immunosuppression, weisolated CD11b⁺ cells from LM-OVA infected tumors and co-cultured themwith in vitro activated CD8 T cells. Indeed, CD11b⁺ cells from LM-OVAinfected tumors were less suppressive to T cell proliferation thanCD11b⁺ cells from uninfected tumors (FIG. 5I), suggesting that Listeriainfection diminishes the immunosuppressive function of myeloid cells andimproves antitumor effector function of CD8 T cells.

More intriguingly, dual-specific CD8 T cells used in ReACT expressedlower levels of several inhibitory receptors (LAG-3, CTLA-4, Tim3 andPD-1) compared to mono-specific CD8 T cells (FIG. 5J), suggesting thatthese reenergized CD8 T cells might be bestowed with enhanced antitumorfunction and less exhausted phenotypes. These results collectivelydemonstrate that intratumoral bacterial infection can largely reversethe immunosuppression in the TME (9,19) and recruit dual-specific CD8 Tcells with greater antitumor properties to the site of tumor.

Discussion

Both adoptive cell transfer of genetically engineered T cells andpathogen-based cancer vaccines are promising strategies to treat cancer.However, adoptively transferred T cells migrate inefficiently to thetumor and readily lose effector function in the immunosuppressive TME.Pathogen-based vaccines can reverse immunosuppression in the tumor, butare less efficient at inducing tumor-specific CD8 T cells with adequatemagnitude and clonal types to confer tumor eradication. In this study,we combined the strength of both approaches and developed an innovativestrategy, ReACT, to treat solid tumors in a preclinical model. ReACTuses a pathogen not only to break the immunosuppressive TME, but also todrive the expansion and migration of tumor-specific T cells to the siteof tumor. We have demonstrated the enhanced antitumor efficacy of thiscombinatorial approach in comparison to either treatment alone inprimary tumor eradication. More importantly, the mice cured from ReACTalso develop immunological memory that protects them from subsequentrechallenge of the same tumor.

To bridge ACT and pathogen-based cancer vaccines together, the inventorgenetically-engineered tumor-specific CD8 T cells with a second TCR thatrecognizes a pathogenic antigen to create dual-specific T cells. Severalstudies have shown that augmented expansion and durability ofdual-specific CD8 T cells clearly increased the antitumor activity andthe overall survival of tumor bearing mice. Nonetheless, tumors were noteradicated in these applications (12,21-23). This is possibly due toinefficient migration of dual-specific T cells to the tumor andunchanged immunosuppressive tumor microenvironment, given that thepathogen was either administrated systemically (23) or not used(12,21,22). In addition, one important distinction of dual-specific Tcell generation in ReACT is to give a pathogen-specific TCR to tumorreactive T cells. This is opposite from previous work that givespathogen (EBV, CMV and Influenza virus) reactive T cells a singletumor-specific TCR (21-23). Our approach allows us to generatepolyclonal dual-specific T cells targeting multiple tumor associateantigens (TAAs) to increase the ability of tumor control.

William Coley was arguably the first to practice cancer immunotherapy acentury ago. Live pathogens have been used as adjuvants (such as BCG) tostimulate patients' immune systems to treat bladder cancer and melanomafor decades (10,11). Pathogen-based immunotherapies induce potent innateimmune responses that break the suppressive tumor microenvironment atleast in part by targeting MDSCs and Tregs (9,19). However, with limitedexpansion of tumor-specific T cells both in quantity and clonal types,the antitumor effects of this approach are transient and rarely able toachieve long lasting antitumor effects (9). New strategies that userecombinant bacteria such as Listeria expressing tumor antigens to treata variety of cancers have shown promising efficacy in clinical trials(9). In this study, we show greater antitumor effects when combiningpathogen-based cancer vaccine with ACT of dual-specific CD8 T cells thanrecombinant Listeria expressing a tumor antigen. This can be explainedby a greater magnitude of clonal expansion of adoptively transferredtumor-specific CD8 T cells than that from endogenous T cells, whichsupports the idea that the initial T cell mediated killing cruciallydepends on sufficiently high doses of T cells within the tumor forsuccessful eradication (24).

In summary, we developed a novel immunotherapy, ReACT, to treat solidtumors and validated its efficacy in proof-of-principle animalexperiments. Given the broad use of both ACT and pathogen-based vaccinesin cancer treatments, this combinatorial strategy holds greattranslational value in treating various malignancies in humans.

Methods

Tumor Cell Lines, Bacteria and Mice

B16-F10, B16-OVA and E0771 were obtained from ATCC and cultured inhigh-glucose DMEM (Cellgro) supplemented with 10% FBS. C57BL/6 mice wereobtained through the National Cancer Institute (NCI) grantees program(Frederick, Md.). Pmel-1 TCR transgenic mice that recognize the MHCclass I (H-2D^(b))-restricted epitope of gp100 presented on the surfaceof B16-F10 melanoma were purchased from Jackson Laboratories (BarHarbor, Mass.). Recombinant Listeria monocytogenes (LM) expressing OVA(LM-OVA) and GP33 (LM-GP33) was developed by Dr. Hao Shen (University ofPennsylvania School of Medicine, Philadelphia, Pa.) and kindly providedby Dr. Susan Kaech (Yale University, New Haven, Conn.)

Tumor Induction and Rechallenge

Melanoma tumors were established by injecting 2×10⁵ B16-F10 cellssubcutaneously (s.c.) on one flank of the C57BL/6 mice, while breasttumors established by injecting at 3×10⁵ cells near the fat pad of thefourth mammary gland in the lower abdomen. Mice that eradicated theirprimary B16-F10 tumors were rechallenged with 1×10⁵ B16-F10 cells on theone flank and 1×10⁵ E0771 cells on the fat pad of the fourth mammarygland from the opposite flank. The eradication of primary tumor wasassessed by no visible and palpable tumor mass at least 6-8 weeks afterthe clearance of tumors following initial treatment. Age- andgender-matched naïve C57BL/6 mice were used as controls. Tumor growthwas monitored by measuring with calipers every other day and tumorvolume was calculated as length×(width)²/2

Retroviral Transductions to Generate Dual-Specific Tumor Reactive TCells and Adoptive T Cells Transfer

To produce retroviral supernatant to express OT-I ovalbumin-specific TCRin T cells, 293T cells were transfected with either MSCV-IRES-GFP (MIG)plasmid, or MIG-OT-I vector along with the pcLEco ecotropic packagingplasmid. At the same time, the splenocytes were harvested from Pmel-1mice and seeded in 24 well plates at 5×10⁶ cells/well and cultured with10 nM gp100 (Genscript) and 10 ng/ml IL-2 (Peprotech) for 24 hours,followed by spinning transduction with prepared retroviral supernatant.After the transduction, these cells were cultured in the original mediumfor another 2 days and washed with PBS. After additional 3 days ofculturing in T cell media containing 10 ng/ml IL-7 and 10 ng/ml IL-15,the positively transduced cells, defined by expression greenfluorescence protein (GFP), were sorted for transfer.

For experiments involving ACT, mice received 5×10⁵ sorted Pmel-1⁺mono-specific or OT-I⁺ Pmel-1⁺ dual-specific CD8 T cells at least sevendays after initial tumor inoculation. At the same time, these mice wereinjected with either 1×10⁴ colony forming unit (CFU) LM-OVA or PBS i.t.

Generation of Polyclonal Tumor Reactive CD8 T Cells

Bone marrow cells were isolated from C57BL/6 mice and cultured in 10%FCS RPMI medium with 200 ng/ml Flt3L for one week. On day 7, DCs wereharvested and incubated with freeze-thawed tumor lysates at a ratio ofone tumor cell equivalent to one DC (i.e., 1:1) as previously described(17). After 18 hours of incubation, DCs were harvested and maturatedwith LPS for 4 hours. The mature DCs and purified CD8 T cells were mixedin 1:2 ratio and cultured together with low dose IL-2 (1 ng/ml) for 24hours. Then the activated CD8 T cells were transduced and sub-culturedas described above.

Immune Cell Isolation from Solid Tumors

The dissected tumor tissues were cut into small pieces and digested with0.7 mg/ml collagenase XI (Sigma-Aldrich) and 30 mg/ml of type IV bovinepancreatic DNase (Sigma-Aldrich) for 45 min at 37° C. The immune cellswere isolated by centrifugation with Lymphocyte Cell Separation Medium(Cedarlane Labs).

MDSC Suppression Assay

As described before (25), splenic CD8 T cells were isolated using theMouse T Cell Isolation Kit (Stem Cell Technology), seeded in 96 wellplates at 2×10⁵ cells/well, and stimulated with anti-CD3 (eBioscience)and anti-CD28 (eBioscience) antibodies. At the same time, the CD11b⁺myeloid cells were sorted from tumors by Fluorescence Activated CellSorting (FACS) and added to these wells at various ratios (1:16, 1:8,1:4 and 1:2). After 48 hours incubation, ³H-Thymidine (1 μCr/well) wasadded and the incubated for 16 hours. Cells were harvested using aPackard Filtermate Harvester 96 and counted by Microbeta counter(PerkinElmer, Beaconsfield, UK).

Statistical Analysis

Graphs were generated and statistical analyses performed using GraphPadPrism version 5.02 (GraphPad Software, Inc.). The overall tumor growthin FIG. 1C was analyzed by one-way ANOVA, while the comparison of tumorfree mice after secondary challenge was determined by Log-rand test. TheKruskal-Wallis with Dunn's multiple comparison test was use to comparethe individual tumor growth curves from different treatment groups. TheSpearman's rank correlation coefficient test was used to determine theassociation between the tumor sizes and cell composition in micereceived different treatments. For all other comparisons, t-tests wereused to determine the statistical significance. *p<0.05; **p<0.01.

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SEQUENCE LISTING STATEMENT

The application includes the sequence listing that is concurrently filedin computer readable form. This sequence listing is incorporated byreference herein.

The invention claimed is:
 1. A purified population of autologousdual-specific lymphocytes which have specificity for two or moreantigens, wherein a population of lymphocytes is isolated from a patientand each lymphocyte expresses an endogenous receptor for a tumorassociated antigen (TAA) and is genetically engineered to express anadditional receptor for a strong antigen derived from a pathogen,wherein the population of dual-specific lymphocytes target a pluralityof TAAs and the strong antigen derived from a pathogen.
 2. The purifiedpopulation of autologous lymphocytes of claim 1, wherein the lymphocytesare selected from the group consisting of CD8+ T cells, CD4+ T cells,and natural killer (NK) cells.
 3. The purified population of claim 1,wherein the autologous lymphocytes are tumor-infiltrating lymphocytes.4. The purified populations of claim 1, wherein the lymphocytes are CD8+T cells.
 5. The purified population of claim 1, wherein the pathogen isselected from the group consisting of listeria monocytogenes, BacillusCalmette-Guérin, tetanus, diphtheria, adenovirus, herpes simplex virus,vaccinia virus, myxoma virus, poliovirus, vesicular stamatis virus,measles virus, influenza virus, and Newcastle disease virus.
 6. Thepurified population of claim 1, wherein the strong antigen is an antigenfrom listeria monocytogenes.
 7. The purified population of claim 1,wherein the lymphocytes are isolated from the tumor of the patient. 8.The purified population of claim 1, wherein the lymphocytes are isolatedfrom the peripheral blood and optionally are PD-1 positive.
 9. Thepurified population of claim 1, wherein the genetically engineeredadditional exogenous receptor is a chimeric receptor.
 10. A compositioncomprising the purified population of dual-specific lymphocytes of claim1 and a pharmaceutically acceptable carrier.
 11. A method of producingan autologous population of dual-specific lymphocytes that can target aplurality of tumor associated antigens and at least one strong antigenderived from a pathogen, the method comprising the steps of: (a)isolating lymphocytes from a patient; (b) purifying the tumor-specificlymphocytes from the isolated lymphocytes; and (c) geneticallyengineering the purified lymphocytes to express a second receptorspecific to a strong antigen derived from a pathogen, wherein theresulting population comprises dual-specific lymphocytes.
 12. The methodof claim 11, wherein step (c) further comprises expanding the isolatedtumor-specific lymphocytes in culture.
 13. The method of claim 11wherein step (a) comprises isolating the lymphocytes from a tumor,peripheral blood, or bone marrow of the patient.
 14. The method of claim11, wherein the pathogen is selected from the group consisting of groupof listeria monocytogenes, Bacillus Calmette-Guérin, tetanus,diphtheria, adenovirus, herpes simplex virus, vaccinia virus, myxomavirus, poliovirus, vesicular stamatis virus, measles virus, influenzavirus, and Newcastle disease virus..
 15. The method of claim 11, whereinthe genetically engineered receptor is a chimeric receptor.
 16. Themethod of claim 11, wherein the lymphocytes are selected from the groupconsisting of CD4+ T cells, CD8+ T cells, and natural killer (NK) cells.17. The method of claim 16, wherein the lymphocytes are CD8+ T cells.18. The method of claim 11, wherein the lymphocytes are stimulated inculture in the presence of an interleukin to stimulate growth.
 19. Themethod of claim 18, wherein the interleukin is selected from the groupconsisting of IL-2, IL-7 and IL-15.
 20. The method of claim 11, whereinthe lymphocytes are stimulated with the strong antigen or TAA in cultureto proliferate.
 21. A method of treating a patient with a tumorcomprising: (a) administering to the patient an effective amount of theautologous dual-specific lymphocytes of claim 1, and (b) injecting thepatient with a strong antigen derived from a pathogen, wherein thestrong antigen derived from a pathogen activates the autologousdual-specific lymphocytes, thereby treating the patient with the tumor.22. The method of claim 21, wherein the autologous dual-specificlymphocytes are injected into the patient intravenously and the strongantigen derived from a pathogen is injected intratumorally.
 23. Themethod of claim 21, wherein the strong antigen derived from a pathogenis selected from the group consisting of a viral antigen and a bacterialantigen.
 24. The method of claim 23, wherein the pathogen is selectedfrom the group consisting of group of listeria monocytogenes, BacillusCalmette-Guérin, tetanus, diphtheria, adenovirus, herpes simplex virus,vaccinia virus, myxoma virus, poliovirus, vesicular stamatis virus,measles virus, influenza virus, and Newcastle disease virus.